Abstract:

The present invention relates to a method preparing co-colloidal
dispersions of active hydrophobic substances, such as pharmaceutical
products, products having cosmetic properties or any other chemical
product. The co-colloidal dispersions thus obtained are characterized in
that they are formed by amphiphilic complexes resulting from combinations
by non-covalent bonds between a hydrophobic active substance and a
suitable hydrophilic molecule.

Claims:

1. Co-colloidal dispersion in an aqueous medium of at least one
supramolecular amphiphilic complex in which the at least one
supramolecular amphiphilic complex comprises at least one hydrophilic
molecule and at least one hydrophobic molecule associated by non-covalent
bonds.

2. Co-colloidal dispersion according to claim 1, wherein the at least one
hydrophilic molecule is chosen from hydrosoluble calixarenes.

3. Co-colloidal dispersion according to claim 1, wherein the at least one
hydrophilic molecule is chosen from anionic hydrosoluble calixarenes.

4. Co-colloidal dispersion according to claim 1, wherein the at least one
hydrophilic molecule of is chosen from parasulfonato calixarenes.

5. Co-colloidal dispersion according to claim 1, wherein the at least one
hydrophobic molecule is chosen from molecules of pharmaceutical interest
and cosmetic substances.

6. Co-colloidal dispersion according to claim 5, wherein the at least one
hydrophobic molecule is chosen from substances that are active on the
peripheral system, substances that are active on the central nervous
system, substances that are active on renal function, substances that are
active on cardiovascular function, substances that are active on
gastro-intestinal function, substances that are active on blood function,
substances that are active on immune function, substances that are active
on hormonal function, substances that are active on genital function,
substances that are active on reproductive functions, anti-inflammatory
substances, anti-parasitic substances, antibiotics, anticancer
substances, antidotes, vitamins, substances for parenteral nutrition,
substances for dermatologic use, substances for topical use, and
substances for ophthalmologic use.

7. A process for dispersion in an aqueous medium of at least one
hydrophobic molecule of pharmaceutical interest comprising forming at
least one supramolecular amphiphilic complex between the at least one
hydrophobic molecule and at least one hydrophilic molecule.

8. A process for dispersion in an aqueous medium of at least one
hydrophobic cosmetic substance comprising forming at least one
supramolecular amphiphilic complex between the at least one substance and
at least one hydrophilic molecule.

9. A process for preparing a co-colloidal dispersion according to claim 1,
comprisingadding a composition comprising, in an organic solvent, at
least one anionic hydrosoluble calixarene to a composition comprising, in
an organic solvent, at least one hydrophobic molecule,adding an aqueous
solvent, andeliminating the organic solvent.

10. Anti-cancer drugs in a form of a co-colloidal dispersion in an aqueous
medium of at least one amphiphilic complex comprising at least one
anionic hydrosoluble calixarene and at least one taxane.

11. Anti-cancer drugs according to claim 10, wherein the at least one
taxane is docetaxel.

12. Anti-cancer drugs according to claim 11, in a form for use by the
intravenous route for the treatment of cancer diseases.

13. Pharmaceutical drugs in a form of a co-colloidal dispersion in an
aqueous medium of at least one amphiphilic complex comprising at least
one anionic hydrosoluble calixarene and thalidomide.

14. Pharmaceutical drugs according to claim 13, in a form for use by the
intravitreal route for the treatment of age-related macular degeneration.

Description:

[0001]The present invention describes a method for dispersing hydrophobic
substances in aqueous phase. Such a method is useful for multiple
applications, notably for the formulation of active pharmaceutical
substances or cosmetics.

[0002]One of the major issues met during the development of biologically
active substances lies in their hydrophobic nature. In aqueous phases,
active substances tend to precipitate, thereby considerably limiting
their bioavailable concentrations and their biological activity. It is
possible to increase the bioavailable concentrations of active
hydrophobic substances by using different means, for example, by
inclusion in suitable molecules, such as cyclodextrins, or by
encapsulation in an suitable colloidal dispersion, such as micelles,
liposomes, or lipidic nanoparticles. The encapsulation methods can also
protect the molecules from degradation by light or by enzymatic
reactions. The other methods that allow modification of the concentration
of active substances are the use of co-solvents or co-solutes or pH
modifications. Besides, the use of co-crystals allows modifications of
the dissolution kinetics and thereby to modify the pharmacokinetics
profile of an active substance.

[0003]To prepare colloidal dispersion, one uses one or several amphiphilic
molecules. An amphiphilic molecule is a molecule that combines covalently
a hydrophobic apolar group and a hydrophilic polar group. Amphiphilic
molecules have a double affinity, for the apolar phases (air, oil,
organic solvents) on one hand, and for water, on the other hand. Lipids
are examples of amphiphilic molecules.

[0004]Above a critical concentration in an aqueous environment,
amphiphilic molecules are generally capable to auto-assemble by forming
colloidal structures that are characterized by particles in suspension.
The type of colloidal structure that is obtained depends on the chemical
structure of the amphiphilic molecules, and notably on the ratio between
the sizes of the polar head and apolar tail of the molecule.

[0005]Colloidal dispersions can be used to increase the bioavailable
concentrations of hydrophobic active substances. The principle is that
the active hydrophobic substance should be associated with the
hydrophobic groups of the amphiphilic molecules.

[0006]However, one drawback of the methods that use amphiphilic molecules
lies in the fact that the maximum percentage (also called charge factor,
calculated in weight units) of active substance that can be dispersed in
the colloidal structure is too low, approximately 5 to 10%. Another
drawback of these colloidal assemblies is their low stability during
time, notably at room temperature.

[0007]These problems have been overcome by using solid lipid nanoparticles
(SLN) that include nanocapsules and polymeric nanospheres. Such
structures are matrix structures. In this case, the charge factors are
generally higher, approximately 30%, and the stability during time is
improved.

[0008]Besides, methods that use calixarenes products have been described.
The US patent 2005/0240051 A 1 authored by N. Yasuda et M. Furukawa
describes a method that uses calixarenes to solubilize carbon-based
materials, such as fullerenes, graphite, diamond and stains, in a
non-aqueous organic solvent such as toluene, an oil or a resin. However,
the calixarenes used in this patent are not soluble in water and this
patent is not related to a dispersion method for pharmaceutical or
cosmetic substances.

[0009]In the patent application WO 03/024583, the authors describe a
system for dispersion in water that uses amphiphilic and non-hydrophilic
calixarenes. These amphiphilic calixarenes can spontaneously
auto-assemble and form colloidal dispersions. The addition of a
hydrophobic molecule to this colloidal calixarenes suspension is possible
but is not necessary.

[0010]In two articles (Eur. J. Pharm. Biopharm., 2004, 58(3), 629-636 and
J. Pharm. Pharmacol., 2004, 56(6), 703-708), W. Yang and M M. De villiers
have shown that it was possible to solubilize nifedipine, furosemide and
niclosamide (active substances of pharmaceutical interest that are poorly
soluble in water) by using para-sulfonato-calixarenes in an aqueous
acidic solution. However, the objectives of this work and the processes
that are used are different from the invention described in the present
patent. Indeed, W. Yang and M M. De villiers describe only a
solubilization method and not a colloidal dispersion method, that is
notably characterized by the presence of particles.

[0011]More recently, the co-crystal systems, based on supramolecular
non-covalent assembly systems, have been used to modify the
pharmacokinetic and physical properties of pharmaceutical products. In
this case, one uses the known non-covalent interactions so that a
crystallizing substance is bound to a pharmaceutical substance, thereby
providing different physical properties, stability and dissolution rate.

[0012]In view of the previous information, the purpose of the invention is
notably to implement a new method that allows the dispersion of
hydrophobic active substances, such as substances of pharmaceutical or
cosmetical interest, in aqueous solution.

[0013]The purpose of the invention is also to propose new dispersions that
do not have the drawbacks mentioned in the previous art and, in
particular, that are stable in time.

[0014]Finally, the purpose of the invention is also to propose new
dispersions which can be used as vehicles for hydrophobic active
substances of pharmaceutical or cosmetic interest.

[0015]In a surprising and advantageous way, the applicant has now just
discovered that these purposes could be reached by implementing a process
of dispersing a hydrophobic molecule, such as a hydrophobic active
pharmaceutical or cosmetic substance of interest, in an aqueous phase,
which includes a step consisting of forming a supramolecular complex
between the said hydrophobic molecule and the said hydrophilic molecule.

[0016]In the following text, the dispersions obtained within the framework
of the present invention are identified by the term "co-colloidal
dispersions."

[0017]So, the process of dispersion according to the present invention,
allows generation of co-colloidal dispersions of hydrophobic active
molecules in aqueous solution.

[0018]Furthermore, these co-colloidal dispersions are stable with time.

[0019]So, in a first version of the invention, a co-colloidal dispersion
in an aqueous medium of at least one supramolecular amphiphilic complex,
in which the said amphiphilic complex includes one hydrophilic molecule
and one hydrophobic molecule that are associated by non-covalent bounds,
is proposed.

[0020]The present invention uses the formation of an inter-molecular and
non-covalent association between a molecule of a hydrophobic active
substance, termed an invited molecule, and an suitable hydrophilic host
molecule. This association results in the formation of supramolecular
amphiphilic complexes combining a polar head and an apolar tail. In
aqueous phases, these supramolecular amphiphilic complexes are capable of
auto-assembling and forming particles suspensions that characterize
colloidal dispersions. The size of the obtained particles ranges between
10 nm and 1 μm, and preferentially between 100 and 450 nm.

[0021]Therefore, this method allows dispersion of a hydrophobic molecule
in an aqueous phase in the form of a dispersion of a colloidal nature. An
aqueous phase contains at least 50% of water and can be constituted, for
example, by pure water, by an isotonic solution, by a physiological
medium or by a pharmaceutical solution for injection or for topical use.

[0022]The co-colloidal dispersions described in the present invention are
different from all the known colloidal dispersions, for example those
that use lipids, because there are no covalent bounds in the assembly of
the amphiphilic entity. They are also different from all the known
colloidal dispersions because the hydrophobic molecule that one wants to
disperse is necessary to form the colloidal structure in the aqueous
medium.

[0023]Thus, contrary to the colloidal dispersions described in the patent
application WO 03/024583, the co-colloidal dispersions described in the
present invention are constituted by the assembly of two molecules, one
being hydrophilic and the other being hydrophobic forming an amphiphilic
supramolecular complex; thus, none of the two molecules can form a
colloidal dispersion when used alone. It is indeed a different state of
matter.

[0024]The host hydrophilic molecules that are used in the invention
include notably products from the calixarene family and preferentially
anionic hydrosoluble calixarenes, for example the para-sulfonato
calix[4]arene and the para-sulfonato calix[6]arene molecules.

[0025]In the sense of the present invention, an hydrophilic substance is a
compound that is soluble in water and that is able to create hydrogen
bonds with water molecules.

[0026]In particular, the calixarenes used in the present invention are
hydrophilic compounds because they bear polar and ionizable functions.

[0027]The calixarenes used in the present invention have the general
structure (I):

##STR00001##

[0028]in which, [0029]R1 represents an hydrogen atom or a polar
group, such as an hydroxyl, a carboxylate, a sulfonate, a phosphonate, a
sulfonamide, or an amide, or an alkyl, alkene or alkyne group, linear,
ramified or cyclic, eventually substituted, notably by a polar group, and
preferentially an hydrogen atom or a sulfonate group. [0030]R2
represents a polar group, such as an hydroxyl, a carboxylate, a
sulfonate, a phosphonate, a sulfonamide, an amide, an ester or an alkyl,
ramified or substituted by a polar group, and preferentially an hydroxyl
if R1 is a sulfonate or a phosphonate group if R1 is an
hydrogen atom. [0031]R3, R4, R5, R6 represent each
and independently an hydrogen atom, an alkyl, alkene or alkyne group,
eventually substituted, notably by a polar group, in particular hydroxyl
functions, substituted or not, and preferentially an hydrogen atom.
[0032]X represents an atom of carbon, (when x=1 and y=1), of sulfur or
oxygen (when x=0 and y=0) or of nitrogen (when x=1 and y=0); [0033]n is a
number between 4 and 8.

[0034]The invited hydrophobic active substances that can be dispersed in
an aqueous phase according to the invention are numerous. The examples
described below show that the invention allows dispersion in the form of
co-colloidal dispersion molecules that have very different chemical
structures. The experiments that were conducted show that the molecules
to be dispersed should present an hydrophobic part and chemical groups
which allow for the non-covalent interactions with the hydrophilic host
molecules. The non-covalent bonds that are implemented can include
hydrogen bonding, ion-ion interactions, ion-dipole interactions,
cation-π interactions, π-π interactions, Van Der Walls forces or
hydrophobic interactions. The size of the hydrophobic active substance is
not a restrictive condition.

[0035]Thus, the purpose of the present invention is also a dispersion
process in an aqueous medium of a hydrophobic molecule of pharmaceutical
interest that comprises a step during which a supramolecular amphiphilic
complex between the said hydrophobic molecule and a hydrophobic molecule
is formed.

[0036]As a variation, a dispersion process in aqueous medium of a
hydrophobic cosmetic substance that comprises a step during which a
supramolecular amphiphilic complex between the said hydrophobic substance
and a hydrophobic molecule is formed.

[0037]The present invention also concerns a process that allows the
obtention of co-colloidal dispersions which consists in adding a
composition comprising, in an organic solvent, at least one anionic
hydrosoluble calixarene to a composition comprising, in an organic
solvent, a hydrophobic molecule, in adding an aqueous solvent, and in
eliminating the said organic solvent.

[0038]In particular, the process allowing the formation of co-colloidal
dispersions is the following.

[0039]1.) Molecules Solubilisation in Organic Solvent.

[0040]The host molecule and the invited molecule are beforehand
solubilized separately in a powerful organic solvent, preferentially
tetrahydrofurane (THF). The products quantities and the solvent volumes
are determined according to the final concentrations and volume that are
wished. For example, at the laboratory scale, to obtain a co-colloidal
dispersion of active substance at a final concentration of 50 mg/L, 2.5
mg of host molecule, preferentially para-sulfonato calix[4]arene, are
solubilized in 5 mL of THF and 2.5 mg of active substance are separately
solubilized in 5 ml of THF.

[0041]2.) Co-Colloid Formation.

[0042]Then, the two solutions are mixed at equal volumes (5 mL for a
preparation at laboratory scale) and maintained under constant agitation,
for example with a magnetic stirrer set at 350 rounds per minute or with
a Vortex type equipment. Then, the final solvent, for example pure water
(50 mL) is progressively added at a fixed rate of 200 mL/s and with
maintaining constant agitation during a half hour.

[0043]3.) Co-Colloid Formation.

[0044]Thereafter, the initial organic solvent is eliminated,
preferentially by evaporation, for example by placing the dispersion
during 15 minutes, under reduced pressure and at a temperature of
40° C. The co-colloidal dispersion that is finally obtained
appears as homogenous, slightly opaque at visual examination, thereby
showing that the hydrophobic active substance is dispersed homogenously.

[0045]The technical conditions (solvent volumes, product quantities,
duration of each phase, etc.) indicated above are given as example and
can, naturally, be adapted according to the nature of the hydrophobic
active substance, as well as the wished final volumes and concentrations.

[0046]The active substances which can be dispersed according to the
invention include for example substances that are active on the
peripheral and central nervous system, on the renal, cardiovascular,
gastro-intestinal, blood, immune, hormonal, genital or reproductive
functions, anti-inflammatory, anti-parasitic, antibiotics, anticancer
products, antidotes, vitamins, products for the parenterale nutrition or
products for dermatologic, topical or ophthalmologic use.

[0048]In the sense of the present invention, a hydrophobic substance is an
active substance whose solubility in water is not sufficient to prepare
formulations at concentrations that are sufficiently high to obtain the
desired activity.

[0049]Preferentially, the hydrophobic active substances of pharmaceutical
interest that can be dispersed as co-colloids include tamoxifen,
tetracaine, chlorhexidine, mifepristone, thalidomide, and the molecules
of the taxane family.

[0050]The taxanes constitute a group of pharmaceutical products, including
notably docetaxel and paclitaxel, that are used for the treatment of
cancer. The taxanes have the property to stop the growth of cancer cells
by interfering with cellular structures, called microtubules, that play
an essential role for cell division.

[0051]Microtubules are formed when cells start to divide and are destroyed
after cell division. Taxanes prevent the microtubules destruction and
thus prevent cell division.

[0052]Docetaxel and paclitaxel are administered to patients by the
intravenous route for the treatment of cancer diseases, notably lung,
prostate, ovary or breast cancers.

[0053]Docetaxel and paclitaxel are molecules with low water solubility.
Thus, the preparation of a pharmaceutical formulation with docetaxel
generally requires to, firstly solubilize docetaxel in a mix of ethanol
and polysorbate, then to dilute this solution in an aqueous solution.
This transparent solution is then injected as an intravenous perfusion.

[0054]However, the presence of residual polysorbate in such a solution can
lead to significant toxicity effects during the administration of this
said solution by the intravenous route. These toxic effects can
necessitate to pre-treat the patients with an anti-inflammatory drug.

[0055]Thus, the process according to the invention presents the advantage
to disperse docetaxel in an aqueous medium without using polysorbate.
Therefore, one obtains a less toxic pharmaceutical formulation that can
be administered by the intravenous route.

[0056]Besides, when the drug under the form of a co-colloidal dispersion
in aqueous medium of at least one amphiphilic complex comprising at least
one anionic hydrosoluble calixarene and docetaxel is administered, this
drug present the additional advantage to produce a satisfactory
anti-cancer effect.

[0057]Therefore, the present invention consists in an anti-cancer drug
under the form of a co-colloidal dispersion in aqueous medium of at least
one amphiphilic complex comprising at least one anionic hydrosoluble
calixarene and one molecule of the taxane family.

[0058]In particular, the purpose of the present invention is a
pharmaceutical drug under the form of a co-colloidal dispersion in
aqueous medium of at least one amphiphilic complex comprising at least
one anionic hydrosoluble calixarene and docetaxel.

[0059]More particularly, the invention relates to a pharmaceutical drug
under the form of a co-colloidal dispersion in aqueous medium of at least
one amphiphilic complex comprising at least one anionic hydrosoluble
calixarene and docetaxel for use by the intravenous route to treat cancer
diseases.

[0060]In particular, this pharmaceutical drug is used to treat breast and
lung cancer.

[0061]Docetaxel can be present in the co-colloidal dispersion at a
concentration ranging from 0.01% to 1% in weight, relatively to the total
weight of the dispersion.

[0062]Besides, thalidomide is a molecule known for its anti-angiogenic
properties. These properties allows the use of thalidomide as a
pharmaceutical drug to treat a specific type of cancer known as multiple
myeloma.

[0063]Thalidomide is a pharmaceutical drug that is generally administered
by the oral route. Besides, there are no pharmaceutical formulations
allowing administration by the parenteral route.

[0064]Thus, the process according to the invention present the advantage
to disperse thalidomide in aqueous medium. Thus, one obtains a
pharmaceutical formulation that can be administered by the parenteral
route, for example by the intravenous, intramuscular, subcutaneous or
intravitreal route.

[0065]Therefore, the purpose of the present invention is also a
pharmaceutical drug under the form of a co-colloidal dispersion in
aqueous medium of at least one amphiphilic complex comprising at least
one anionic hydrosoluble calixarene and thalidomide.

[0066]In particular, this pharmaceutical drug can be administered to
cancer patients and for whom the oral administration is difficult or
impossible.

[0067]Indeed, some patients present with lesions of the buccopharyngeal
region or the oesophagus that make the oral administration of
pharmaceutical drugs very difficult. In this case, thalidomide can be
administered by the parenteral route, and notably by the intravenous
route.

[0068]In particular, thalidomide can be administered to patients by the
intravitreal route to treat age-related macular degeneration (AMD.)

[0069]AMD is disease of the retina that is caused by a progressive
degeneration of the macula, the central part of the retina, that appears
most often from the age of 50 years, and more frequently from the age of
65 years, provoking an important decline of visual capacity, but not a
complete loss.

[0070]AMD is characterized by the appearance of choroidal neovessels (new
blood vessels). They develop either under the pigmented epithelium and
thus are designated as "occult" or above the epithelium and thus are
designated as "visible." Because of its anti-angiogenic properties,
thalidomide can prevent the development of new vessels and thus stabilize
the progression of the disease.

[0071]The present invention relates more particularly to a pharmaceutical
drug under the form of a co-colloidal dispersion in aqueous medium of at
least one amphiphilic complex comprising at least one anionic
hydrosoluble calixarene and thalidomide for its use by the intravitreal
route to treat age-related macular degeneration.

[0072]Thalidomide can be present in the co-colloidal dispersion at
concentrations ranging from 0.01% to 1% in weight, relatively to the
total weight of the dispersion.

[0073]Co-colloidal dispersions can also contain miscellaneous additives
such as osmotic pressure regulators, for example sucrose or glycerine,
oxidants such as alpha-tocopherol or ascorbic acid or preservatives such
as methyl, ethyl- and butyl-paraben.

[0074]The compositions for cosmetic use prepared according to the
invention include preparations for skin or hair, such as shampoos,
preparation for use on skin or lotions for sun protection.

[0075]The following examples are intended to better understand the
invention without presenting a limitating character. These examples are
illustrated by FIGS. 1-4 in annex that present co-colloidal dispersions
which are consistent with the present invention and which were
characterized by means of various analysis techniques.

EXAMPLES

Preparation of Co-Colloidal Dispersions

[0076]The examples described below allowed to characterize the method of
preparation of the co-colloidal dispersions as well as to characterize
the quality the said dispersions.

[0077]One prepares a co-colloidal dispersion according to the
above-mentioned process. Thus, in a first solution, 2.5 mg of
para-sulfonato calix[4]arene (designated as C4S in the tables below) are
solubilized in 5 mL of THF and, in a second solution, 2.5 mg of tamoxifen
(anti-cancer drug) are solubilized separately in 5 mL of THF. Tamoxifen
is a hydrophobic active substance.

[0078]Thereafter, the two solutions are mixed at equal volumes and
maintained under constant agitation with a magnetic stirrer set at 350
rounds per minutes or a Vortex type equipment. Then, 50 mL of pure water
are progressively added at a fixed rate of 200 mL/s while maintaining
constant agitation during a half hour.

[0079]Thereafter, THF is evaporated by placing the mixed solution during
15 minutes, under reduced pressure and at a temperature of 40° C.

[0080]Following this process, a co-colloidal dispersion is obtained with a
final concentration of tamoxifen and C4S of 50 mg/mL. The co-colloidal
dispersion appears homogenous at visual examination, thereby indicating
that tamoxifen was dispersed homogenously.

[0081]This process was done three times by using para-sulfonato
calix[6]arene (designated as C6S in the tables below) and tamoxifen at
different concentrations, so as to obtain co-colloidal dispersions with
different final concentrations of para-sulfonato-calix[6]arene and
tamoxifen.

[0082]This process was also done four times with using para-sulfonato
calix[4]arene and tetracaine at different concentration so as to obtain
final co-colloidal dispersions having different final concentrations of
para-sulfonato calix[4]arene and tetracaine (local anesthetic).

[0083]This process was done twice with using para-sulfonato calix[6]arene
and para-sulfonato calix[4]arene with chlorhexidine (antibiotics) at
similar concentrations.

[0084]This process was done twice with using para-sulfonato calix[6]arene
and para-sulfonato calix[4]arene with mifepristone (abortifacient) at
similar concentrations.

[0085]Tamoxifen, tetracaine, chlorhexidine and mifepristone used in these
preparations are pharmacologically-active substances known for their weak
solubility in water.

[0086]The following table indicates the co-colloidal dispersions that were
obtained, as well as the final concentrations in host molecules and in
hydrophobic active substances (invited molecules) present in these
dispersions.

[0087]In all cases, a homogenous, slightly white, co-colloidal dispersion
was obtained. Thus, the hydrophobic active molecules were dispersed in an
aqueous phase while they are little or weakly soluble in water. Using
this method, these co-colloidal dispersions can be used as vehicles for
active hydrophobic substances of pharmaceutical interest.

[0088]Different techniques were then used to characterize a co-colloidal
dispersion having a final concentration in tamoxifen and in
para-sulfonato calix[6]arene equal to 50 mg/mL (corresponding to the
example no. 2 in the above table). In particular, dynamic light
scattering, atomic force microscopy, scanning electronic microscopy and
transmission electronic microscopy are used. These techniques allow the
analysis of the particles in suspension in aqueous phase or after drying.

[0089]Dynamic Light Scattering (DLS) allows the obtention of information
regarding article sizes. When a monochromatic and polarized light beam
hits a particle, the light is diffused in all directions in space. The
variations in intensity of the diffused light are associated with the
diffusion rate of the molecules in the studied region, because they are
animated by brownian movements. Data are directly analyzed to provide
diffusion coefficients. When several molecular species are present, a
distribution of diffusion coefficients can be observed. Thereafter, these
data are treated to obtain the particles diameters. Indeed, the relation
between the diffusion coefficient and the size is based on the
theoretical relations of the brownian movements of spherical particles
(Stockes-Einstein law). Thus, for a medium with known viscosity, the
measure of the distribution coefficients is sufficient to calculate the
particles hydrodynamic radius. The measurement of the size of particles
in solution is done at room temperature. The analyses are carried out
using a 4700C MALVERN spectrometer. The light source is a SIEMENS 40 MW
laser. Each measurement is repeated ten times, the sizes and the reported
polydispersity indices correspond to the mean of the ten measurements.

[0090]Observation by Atomic Force Microscopy (AFM) consists in a surface
analysis, by mean of a very fine point of a few micrometers long and one
hundred of Angstroms of diameter, that is set up at the extremity of
mobile arm made of silicium, called a microcantilever, and having a known
force. The different marketed equipments present with various geometries.
The AFM that was used is based on the Explorer technique (Topometrix
Inc.). In this set up, the piezo-electrical ceramics that is used to
generate the scanning movement holds the micro-cantilever. With the other
techniques, that are more commonly used, the ceramics holds the sample
and the AFM head stays without movements. The equilibrium of forces
between the surface of the sample and the point induces modifications in
the positions of the microcantilever. The signal is recorded on a
photo-detector with four quarters via the reflexion of a laser beam on
the microcantilever which deflection is proportional to the forces acting
on the probe and is thus measured. Thereafter, these data are transformed
into spatial coordinates, thereby generating a surface image. AFM
measures the forces between the point and the sample. These forces depend
on the nature of the sample and on the distance between the point and the
sample. When the point approaches the surface, it is submitted firstly to
forces that are attractive at long distances (van der Waals forces).
Thereafter, when approaching more the surface, the electronic orbitals of
the point and of the sample generate repulsive forces that neutralize the
attractive forces before becoming the dominant forces. The measurements
are done by using an "Explorer ThermoMicroscope" microscope that is
equipped with a 100 μm scanner in non-contact mode. The scanning speed
is 1-2 Hz. For each sample, the scannings are done at 50 μm, 20 μm,
10 μm and 5 μm. The microcantilevers are made of silicium, the
resonance frequency f0 is 260 kHz and the stiffness constant is 45
N.

[0091]For the studies done with scanning electronic microscopy, a 50 μL
sample of co-colloidal dispersion is placed on glass strip, then the
sample is allowed to dry at room temperature during 18 hours. The samples
are then covered with a gold-palladium layer and observed with a Hitachi
S800 electronic microscope at 15 kV.

[0092]Some studies that use transmission electronic microscopy after
freeze fracture and after negative staining are done. For these studies,
a 5 μL sample of co-colloidal dispersion at 0.2 mM on a copper grid
(300-mesh) covered with a Formvar® film (Polyvinyl formal). After 5
minutes adsorption, the samples are negatively stained, either with a
sodium silicotungstate aqueous solution at 1%, or with an uranyl acetate
solution at 4%. They are immediately observed with a Philips CM120
electronic microscope at 80 kV.

[0093]The co-colloidal dispersion according to example no. 2 was studied
as follows:

[0094]Atomic Force Microscopy (AFM) Observation

[0095]FIG. 1 shows the image obtained in microscopy of a co-colloidal
dispersion that was made with tamoxifen and para-sulfonato calix[6]arene
C6S according to example no. 2.

[0096]Thus, the AFM studies show the existence of nanoparticles, even
after drying of the sample.

[0097]A topographical analysis of the size of these particles reveals an
height of 75 nm and a diameter of approximately 300 nm. This diameter is
slightly greater that the diameter determined by dynamic light scattering
diffusion (see results above) which is explained by the slight flattening
of particles after drying of the sample

[0098]Transmission Electronic Microscopy Observations

[0099]FIG. 2 shows images of a co-colloidal dispersion according to
example no. 2 using scanning electronic microscopy with three different
magnifications.

[0100]FIG. 3 shows images of a co-colloidal dispersion according to the
example no. 2 using transmission electronic microscopy after negative
staining with three different magnifications.

[0101]FIG. 4 shows images of a co-colloidal dispersion made with tamoxifen
and para-sulfonato calix[6]arene using transmission electronic microscopy
after freeze drying fracture and with three different magnifications.

[0102]These images allow identification the particles in the co-colloidal
dispersion. They show notably a spherical structure, but also an internal
structure organized in small vesicles. Such a structure is different from
the multi-lamellar structure of liposomes.

[0103]Besides, similar images were obtained with co-colloidal dispersions
made with para-sulfonato calix[6]arene and griseofulvin or chlorhexidine.

[0104]Measurement of the Particles Size and Polydispersity

[0105]The co-colloidal dispersion made with tamoxifen and para-sulphonato
calix[6]arene according to the example no. 2 was analyzed using dynamic
light scattering immediately after its formation.

[0106]The measurements were repeated ten times and show the existence of a
co-colloidal dispersion constituted with mono-dispersed particles having
a mean diameter of approximately 230 nm. The polydispersity index is 0.03
thereby demonstrating that the particles have a constant diameter.

[0107]These measurements were repeated ten times on the co-colloidal
dispersion according to the examples 1 and 3 to 12. The results are
grouped in the following table.

[0108]These results show that the preparation method for co-colloidal
dispersions allow for obtaining populations of particles that are
relatively homogenous with sizes ranging approximately between 150 and
450 nm, according to the nature of the host molecules and of the invited
molecules and of the concentrations ratio between the two molecules.

[0109]Stability Study of Co-Colloidal Dispersions

[0110]The stability of co-colloidal dispersions according to the examples
1 to 12 was studied for temperatures of 4, 20, 40 and 80° C. The
dispersions are placed in incubators with controlled temperatures and
samples are periodically taken to analyze the size and the polydispersity
of the particles or the morphology by atomic force microscopy. The
results are grouped in the following table.

TABLE-US-00003
Dispersion Host molecule Particles stability at different temperatures as
no. Invited molecule measured by dynamic light scattering
1 C4S (50 mg/l) At 20° C., the mean particle size slightly
decreases from 190
Tamoxifen (50 mg/l) to 130 nm over a 15-day period. At 80° C., the
particles
mean size decreases from 190 to 175 nm over a 15-day
period
2 C6S (50 mg/ml) At 4° C., 20, 40 and 80° C., the mean
particle size is
Tamoxifen (50 mg/l) maintained close to 200 nm over a 43-day period
5 C4S (50 mg/l) At 4° C., the mean particle size increases from 205
to 230 nm
Tetracain (50 mg/l) and over a 15-day period, then is maintained stable
during 28 days. At 20, 40 and 80° C., the mean particle size
decreases from 205 to 165 nm and over a 15-day period,
then is maintained stable during 28 days.
8 C4S (50 mg/l) At 4 and 20° C., the mean particle size decreases
from 220 to
Tetracaine (50 mg/l) 179 and 150 nm, respectively, over a 15-day period
11 C4S (50 mg/l) At 20 and 80° C., the mean particle size decreases
from 238
Mifepristone (50 mg/l) to 220 nm over a 15-day period
12 C6S (50 mg/l) At 20° C., the mean particle size decreases from
190 to 174 nm
Mifepristone (50 mg/l) over a 15-day period. At 80° C., the mean
particle size is
maintained at 190 nm over 15 days.

[0111]These results show that the particle size varies slightly, even when
the preparations are maintained during several weeks at low temperatures
(4° C.) or high temperatures (80° C.), thereby showing the
remarkable stability of co-colloidal dispersions.

[0112]Thus, the results show that the particle size is almost uninfluenced
by the temperature used for storage. Similar results were observed for
other co-colloidal dispersions that are not shown as examples.

[0113]Study of the Interactions Between Co-Colloidal Dispersions and
Albumin

[0114]In order to envisage an intravenous administration of co-colloidal
dispersions, it is necessary to ensure that such dispersions do not
aggregate when in contact with albumin, which is the most abundant
protein in the blood system. Thus, the interaction of co-colloidal
dispersions with albumin was also studied. A solution of bovine serum
albumin (BSA) at 1 mg/mL was added to a co-colloidal dispersion made with
tamoxifen and the mixed solution was analyzed by dynamic light scattering
and by atomic force microscopy.

[0115]The analyses by light scattering show that the particles size with
BSA are greater than 1 μm, but they stay monodispersed. This
phenomenon can be explained by the presence of a protective matrix of
proteins that surrounds the colloidal particles. On the contrary, the
images obtained by atomic force microscopy reveal the existence of
particles with sizes that are much smaller than the normal particles size
(15 nm instead of 200 nm or 1000 nm). This can be explained by the fact
that BSA forms a relatively dense protein gel that covers the
co-colloidal particles and let see only a part of the particles at the
surface of the area examined by atomic force microscopy. Thus, the
interactions with the major blood protein do not change the co-colloidal
systems, but albumin can form a matrix around the particles, which can
protect them during transport in blood.

[0116]Example of Preparation of a Co-Colloidal Dispersion Comprising
Anti-Cancer Drugs

[0117]Docetaxel, azacytidine and thalidomide are pharmacologically-active
substances, known for their poor solubility in water and known for their
therapeutic activity in certain types of cancer. Co-colloidal dispersions
have been prepared according to the above-mentioned process and stored
during one week at room temperature. The average particle size has been
measured by DLS according to the above-mentioned method.

[0121]When the tumor reaches a volume of 100 to 200 mm3, the animals
are randomly allocated to treatment groups. According to their group, the
animals receive a series of intravenous injections of either placebo or
docetaxel formulated under the form of a co-colloidal dispersion at
concentrations ranging from 0.01% to 1%. The animals are examined daily
for clinical signs and the tumor volume is measured. A satisfactory
anti-cancer effect was observed with the treatment with docetaxel
formulated under the form of co-colloids.

[0122]Application of Thalidomide Formulated as Co-Colloids to Treatment of
AMD

[0123]Rabbits (strain: Fauve de Bourgogne) are used to induce a choroidal
neovascularization. Burns of approximately 75 μm area are induced in
the right eye of the animals with an argon laser at 532 nm applied for
0.1 second with a 150 mW intensity and using a Viridis photocoagulator
(Quante Medical) around the optical disc and between the main vessel
branches.

[0124]These burns induce the formation of neovessels that are measured by
periodical opthalmological examinations. The animals are randomly
allocated to treatment groups. According to their group, the animals
receive an intravitreal injection of placebo or thalidomide formulated
under the form of co-colloids at concentrations ranging from 0.01% to 1%.
A satisfactory inhibitory effect of neovessels development is observed in
the animals treated with thalidomide formulated under the form of
co-colloids.